Structural Studies of Alix and ESCRT Complexes in HIV-1 Budding HIV-1 particle assembly and release depend on a protein network that includes Alix and Vps4A/B, and four multiprotein complexes: Hrs/STAM and ESCRT-I, II, and III. These proteins and complexes are conserved from yeast to human, and their normal function is to sort monoubiquitinated receptors, enzymes, and other cargo to the lysosome or vacuole. Inactivation of any one of several proteins tested in this network blocks HIV release and infectivity. The objectives of this projects are to 1) map the molecular interactions between HIV and ESCRT components at the level of individual protein domains;2) characterize the binding affinities and relevant structures of the components involved in these interactions;and 3) in collaboration with Eric Freed, NCI, to design means for inhibiting HIV-1 budding from cells. The major role of the ESCRTs in the budding of enveloped viruses, including HIV-1, is thought to be the scission of the neck connecting the plasma membrane to the nascent virion. Cleavage of this neck is thought to be carried out by the same mechanism in multivesicular body biogenesis and in cytokinesis. It has been thought that the ESCRT-III complex was most likely involved in the process, but direct evidence was lacking. In yeast, ESCRT-III consists of Vps20, Snf7, Vps24, and Vps2, which assemble in that order, and require the ATPase Vps4 for their disassembly. The ESCRT-III-dependent budding and scission of intralumenal vesicles into giant unilamellar vesicles was reconstituted and visualized by fluorescence microscopy. Three subunits of ESCRT-III, Vps20, Snf7, and Vps24, were sufficient to detach intralumenal vesicles. Vps2, the ESCRT-III subunit responsible for recruiting Vps4, and the ATPase activity of Vps4 were required for ESCRT-III recycling and supported additional rounds of budding. The minimum set of ESCRT-III and Vps4 proteins capable of multiple cycles of vesicle detachment corresponds to the ancient set of ESCRT proteins conserved from archaea to animals.